INTRODUCTION
Corynebacterium ulcerans is widely distributed in the environment, and it is considered a commensal bacterial species in domestic and wild animals. It is known that this bacterium can cause cutaneous inflammation including mastitis in dairy cows [Reference Fox and Frost1–Reference Tejedor3]. Recently, the microorganism has been increasingly recognized as an emerging zoonotic agent of diphtheria-like illness. In humans, diphtheria is an upper respiratory tract illness caused by C. diphtheria, and it is characterized by sore throat, low-grade fever, and the formation of a pseudomembrane on the tonsils, pharynx, and/or nasal cavity [Reference Murphy and Baron4]. However, C. ulcerans has been detected in the pharynx of patients with diphtheria-like symptoms, and the isolates appear to produce diphtheria toxin (this type of C. ulcerans is referred as toxigenic C. ulcerans) [Reference Mikhailovich5–Reference De Zoysa7]. The toxigenic C. ulcerans strains harbour lysogenic beta-corynephages bearing the tox gene, which encodes the diphtheria toxin, and are thus responsible for the systemic symptoms of diphtheria [Reference Sing8]. In recent epidemiological surveys, toxigenic C. ulcerans was isolated from companion animals such as dogs and cats, and livestock such as cows and sheep; thus, these animals were strongly implicated as sources of human infections [Reference De Zoysa7, Reference Lartigue9, Reference Sykes10].
Twelve cases of diphtheria or diphtheria-like disease associated with toxigenic C. ulcerans have been reported in Japan since toxigenic C. ulcerans was first detected in 2001 [Reference Hatanaka11–Reference Yoshimura, Yamamoto and Komiya17]. In several cases, patients were confirmed as having had direct contact with dogs or cats with/without dermatitis or respiratory symptoms such as rhinitis [Reference Hatanaka11, Reference Yoshimura, Yamamoto and Komiya17]. The genotypes of toxigenic C. ulcerans isolates from patients were identical to those of the stray cats that they had physical contact with [Reference Noguchi15]. More recently, toxigenic C. ulcerans was detected in domestic dogs and cats in Japan via molecular approaches [Reference Yoshimura, Yamamoto and Komiya17, Reference Katsukawa18]. In Japan, people generally have close contact with these companion animals more frequently than livestock and share living environments with their pets. In veterinary hospitals, clinical symptoms of rhinitis are frequently identified in cats; however, infection with C. ulcerans in these pets, particularly infection by toxigenic C. ulcerans, remains unclear. In the present study, we investigated C. ulcerans infection in pet cats with clinical symptoms and characterized the isolates, including possession of the tox gene, to assess the potential risk to humans.
MATERIALS AND METHODS
Animals
In total, 37 cats that were brought to animal hospitals (16 hospitals) in Osaka, Japan between October 2009 and March 2010 were included in the study. The animals displayed chronic rhinitis (Table 1) and their nasal discharge or rhinal secretions were collected using a cotton swab (Seedswab no. 2, Eiken Chemical Co. Ltd, Japan) for examination.
Table 1. Information on cats with Corynebacterium ulcerans isolated and summary of clinical findings
FIV, Feline immunodeficiency virus, FeLV, feline leukaemia virus.
Isolation of bacteria
To isolate bacteria for 48 h, sampled swabs were cultured on sheep blood agar and selective medium (Katsukawa medium) at 35 °C, 5% CO2 [Reference Katsukawa18]. Katsukawa medium consists of heart infusion agar, 0·03% potassium tellurite (w/v), 10% sheep blood (v/v), and 0·05% activated charcoal (w/v) [Reference Katsukawa18]. Samples were incubated for 18–24 h, after which colonies were transferred to dextrose sucrose starch (DSS) agar medium at 24, 30, and 48 h [Reference Jacherts19] to assess glucose and sucrose fermentation. The isolates, which were positive for glucose fermentation and negative for sucrose fermentation, were then characterized by Gram staining and evaluated using the catalase and urease tests. All Gram-positive organisms that were positive for catalase and urease production were suspected to be C. ulcerans. The isolates were biotyped using an API Coryne kit (bioMérieux, France) according to the manufacturer's instructions.
Detection of tox gene by polymerase chain reaction (PCR)
To detect the tox gene in C. ulcerans, bacterial DNA templates were purified from the colonies and PCR analysis was performed as described previously [Reference Mikhailovich5, Reference Hirai-Yuki20, Reference Katsukawa21]. The resulting product were purified and directly sequenced on an automated sequencer (PRISM 3100 Genetic Analyzer, Applied Biosystems, Japan) using the primer pairs as described above.
Elek immunoprecipitation test
The Elek immunoprecipitation test was performed using the antitoxin-in-well procedure to determine the exotoxin production of the isolates as described previously [Reference Hirai-Yuki20]. In brief, a central well (5 mm diameter) was prepared in an agar plate using a sterile stainless-steel blade followed by aspiration of the excised agar. The well was filled with 9 μl of standard diphtheria antitoxin (4·5 IU) (kindly provided by the National Institute of Infectious Diseases, Japan) and then surrounded by C. ulcerans strains that were stabbed into the agar at a distance of 10 mm from the edge of the centre well. C. diphtheriae PW8 was used as a positive control. The precipitation lines formed were observed after incubation for 1–2 days at 37 °C.
Cytotoxicity assay for diphtheria toxin
Cytotoxicity assays for diphtheria toxin were performed using Vero cells to determine the exotoxin production of the isolates as described previously [Reference Hirai-Yuki20]. In brief, the liquid that condensed at the base of the Loeffler slant (Loeffler condensation) was collected after cultivating the C. ulcerans isolates for 3 days. The samples were filtered, and aliquots (25 μl) of the sample were added to Vero cells seeded in culture plates. The cytotoxic effect was determined 4 days after inoculation. To confirm the cytotoxic effect of diphtheria toxin, aliquots were mixed with a standard antitoxin (1 IU/ml) and added to Vero cell cultures after incubation at 37 °C for 30 min. The neutralization endpoints were determined 4 days after inoculation.
Antibiotic susceptibility testing
Antibiotic susceptibility tests were conducted by the broth microdilution method using a Dry Plate DP24 (Eiken Chemical Co. Ltd). We used the following 16 antibiotics: benzylpenicillin (PCG), ampicillin (ABPC), cefazolin (CEZ), cefotiam (CTM), cefotaxime (CTX), cefaclor (CCL), cefditoren (CDTR), flomoxef (FMOX), imipenem (IPM), meropenem (MEPM), erythromycin (EM), clindamycin (CLDM), minocycline (MINO), vancomycin (VCM), levofloxacin (LVFX), and sulfamethoxazole–trimethoprim (ST). Sensitivity was assessed according to the Clinical and Laboratory Standards Institute's (CLSI) standard (M45-A) for Corynebacterium species. The sensitivity of the isolates to the eight drugs, for which CLSI standard values were not available was referenced from the data for similar drugs as follows: PCG was a reference for ABPC; CTX was a reference for CEZ, CTM, CCL, CDTR, and FMOX; TC was a reference for MINO; and CPFX was a reference for LVFX.
RESULTS
C. ulcerans isolates were detected in two of the 37 domestic cats. The infected cats were relatively young, one was a 3-year-old male (no. 7) and the other was a 2-year-old female (no. 32). The isolates showed similar characteristics to those of C. ulcerans, including growth in sheep blood agar and Katsukawa medium, and DSS medium and positivity for API Coryne tests. Consequently, these isolates designated 0011326 and 0111326 as profiling numbers were identified as C. ulcerans. They were identified as toxigenic C. ulcerans by PCR, Elek test, and cytotoxicity assay (Table 2). The 2009-81 and 2010-18 isolates were susceptible to PCG, ABPC, CEZ, CTM, CTX, CCL, CDTR, FMOX, IPM, MEPM, EM, MINO, VCM, LVFX, and ST. The 2010-18 isolate was found to be resistant to CLDM (Table 3). The infected cats were kept indoors; however, they were allowed to roam freely outside (Table 4).
Table 2. Characteristics of Corynebacterium ulcerans isolates
DSS, Dextrose sucrose starch; PCR, polymerase chain reaction.
Table 3. Antibiotic susceptiblity findings for Corynebacterium ulcerans (broth microdilution method)
a Assessments of sensitivities were determined according to the Clinical and Laboratory Standards Institute's (CLSI) standard (M45-A) for Corynebacterium spp.
b Antibiotic susceptibility tests were conducted by the broth microdilution method using a Dry Plate DP24 (Eiken Chemical Co. Ltd).
c Criteria for Staphylococcus spp.
Table 4. Summary of rearing environments of examined cats
DISCUSSION
In the present study, we surveyed 37 domestic cats and detected toxigenic C. ulcerans from two animals. This result, in addition to previous findings, suggests that toxigenic C. ulcerans is distributed over a wide area in Japan [Reference Hirose12–Reference Nakada14, Reference Ogawa22]. In a cat infected with C. ulcerans, treatment with a steroid followed by antibiotics showed improvement. The isolates detected in the present study showed susceptibility to most of the drugs by antibiotic susceptibility testing, including antibiotics being used as treatments for the cats; however, drug treatments alone were insufficient for improvement of symptoms. This steroid appears effective for treating severe nasal obstruction, as reported in a previous study [Reference Ogawa22]. It is believed that improvement in this disease may be associated with other factors in cats such as the presence of another disease which decreases immunity or toxin production. Our results regarding antibiotic characteristics of the isolates, which had susceptibility to most of the drugs or CLDM only, are in agreement with previous studies [Reference Nakada14, Reference Ogawa22]. In addition, Corynebacterium spp. can be frequently isolated from the external ear or skin surface, and also from the nasal cavity of cats without any inflammation [Reference Nakazima26]. Therefore, there is a possibility that C. ulcerans could exist as part of the normal bacterial flora in cats, and the prevalence of toxigenic C. ulcerans is a result of differences in the environment or maintenance of the animals. Thus, epidemiological research is needed to clarify these issues in the future. In previous surveys it was reported that most infected cats were allowed to roam freely, or were stray animals. Our results are congruent with previous findings, and thus the infection can be due to activity outside of the home.
In our study, owners who kept the infected cats did not display any clinical symptoms, but cases of owners acquiring infections from their cats have been reported [Reference Noguchi15, Reference Yoshimura, Yamamoto and Komiya17, Reference Hatanaka23–Reference Taylor25]. In some reports, owners with a weakened immune system, even if they did not have a disease, could be infected by C. ulcerans. In Japan, most infected people were aged >50 years, and these findings may be related to their antibody titres against diphtheria [Reference Kamada24]. In other countries, injection of diphtheria toxoid could decrease the severity of infection by toxigenic C. ulcerans [Reference Taylor25]. It is thought that humans can generally be infected due to nasal catarrh and in some cases due to abscess or eye mucus [Reference Yoshimura, Yamamoto and Komiya17, Reference Nakazima26]. Therefore, it is necessary to investigate various bacterial infectious diseases in cats.
The number of patients with diphtheria in Japan reached 86000 in 1945. After that point infections decreased, and there have been no reports since, except for one case reported in 1999 [27]. The Ministry of Health, Labour and Welfare reported that effective vaccination enables infants to possess high levels of antibodies against diphtheria leading to a marked decrease in antibody prevalence rates in adults aged >45 years [27]. Effectiveness of vaccination for diphtheria has not been fully evaluated [Reference Urakawa16, Reference Schuhegger, Kulgler and Sing28, Reference Dias29], and thus further epidemiological surveys of toxigenic C. ulcerans must be conducted to solve this controversial issue. Veterinarians should consider infection with C. ulcerans in cats with purulent disease, particularly upper respiratory tract diseases such as nasal catarrh. However, there are no inspection agencies in Japan to identify C. ulcerans or assess its toxin production. Therefore, it appears important to ensure there is cooperation between clinical sites and research institutions or develop simple assay methods. In the present study, we identified C. ulcerans by API Coryne. More recently, new methods such as partial RNA polymerase b-subunit (rpoB) sequencing or matrix-assisted laser desorption-ionization–time-of-flight mass spectroscopy (MALDI–TOF/MS) have been reported [Reference Khamis, Raoult and La Scola30, Reference Marini31]. For further analysis, these highly sensitive identifications could be useful in helping to control the infection.
In humans, animals play an important role in the community, and they are closely associated with human society. The risk of zoonosis from domestic animals to humans such as infection by Brucella canis [Reference Lucero32] and Capnocytophaga canimorsus [Reference Gaastra and Lipman33], in addition to toxigenic C. ulcerans, is increasing, but the pathogenicity of most microorganisms in animals remains unclear. Thus, veterinarians may struggle to explain the risks to owners or to treat the animals. In this study, we isolated toxigenic C. ulcerans from domestic cats with chronic rhinitis, and it is believed that this bacterium can cause respiratory tract infection in cats. The infected cats were kept indoors or allowed to roam freely outside, and they were probably in close contact with their owners. Thus, diseases generally found in popular animals could be contracted by humans.
Thus, systems for correct diagnosis and detailed examination of diseases are needed, and people must be educated to develop a healthy relationship between humans and animals. Furthermore, it is important to devise inspection systems or conduct epidemiological surveys in cooperation with veterinarians in animal hospitals and public health officials.
ACKNOWLEDGEMENTS
The authors thank Dr Motohide Takahashi and Dr Takako Komiya, National Institute of Infectious Diseases for effective advice. This investigation was conducted with the cooperation of the Osaka Prefecture Veterinary Medical Association as part of a research activity by a task force on relief activities for animals in disasters. The authors thank the staff members of Ishizuka Animal Hospital (Kisiwada City), Izumi Animal Hospital (Izumi City), Aim Pet Clonic (Izumiotsu City), Oosita Animal Hospital (Sakai City), Kitashin Animal Hosipital (Izumi City), Kitasenri Animal Hospital (Minoh City), Cookie Animal Hospital (Sakai City), Satsukidai Animal Hospital (Kisiwada City), Satou Animal Hospital (Kisiwada City), Sippo Animal Hospital (Izumi City), Tanpopo Animal Hospital (Kaizuka City), Tsuruyamadai Animal Hospital (Izumi City), Hirano Animal Hospital (Izumiotsu City), Matsuoka Animal Hospital (Hannan City) and Leaf Animal Hospital (Kaizuka City) at Osaka Prefecture, Japan for the samples. This work was supported in part by Japan Science and Technology Agency, A-STEP No.4442 (to K.S., H.T., M.F., M.M.). The authors thank Enago (www.enago.jp) for the English-language review.
DECLARATION OF INTEREST
None.
